Antilipidemic methods using glutamic acid,threonine and proline

ABSTRACT

METHODS OF PRODUCING HYPOLIPIDEMIC ACTIVITY IN SUBJECTS HAVING ABNORMALLY HIGH LEVELS OF LIPIDS (CHOLESTEROL) IN THE BLOOD STREAM USING A COMBINATION OF GLUTAMIC ACID AND THREONINE ADMINISTERED INTERNALLY IN QUANTITIES GREATER THAN PRESENT IN THE SUBJECT&#39;&#39;S NORMAL DIET. PROLINE MAY ALSO BE ADDED.

United States Patent 3,574,857 ANTILIPIDEMIC METHODS USING GLUTAMICACID, THREONINE AND PROLINE William H. Cevallos, Devon, Pa., assignor toSmith Kline & French Laboratories, Philadelphia, Pa.

orally or parenterally, to animals in amounts greater than those in thenormal diet. An equivalent amount of either amino acid alone does notgive a statistically significant eifect.

No Drawing Filed Apr 1 1968 Sen 717 943 5 Additionally, I have foundthat combinations of glu- Int CL A tarnic acid, threonine and proline aswell as glutamic Us, 424 319 5 Cl i acid, threonine, proline, alanineand glycine also have similar significant hypolipidemic effects inanimals. The three acid combination is especially preferred since itgives ABSTRACT OF THE DISCLOSURE 10 hypolipidemic effects equivalent tothe five-component tu e,

Methods of producing hypolipidemic activity in submlX jects havingabnormally high levels of lipids (cholesterol) i fi zi gfi g i g gg ggggi g g g i g in the blood stream using a combination of glutamic acid0 both Si nificagt criteria of h and threonine administered internallyin quantities greater F es g yp 15 11 rdemic activit Since the liver isthe site of the catathan resent 1n the sweets normal diet Prohne mayalso p Y b 5 d bOllSIIl of cholesterol, lowering the cholesterol levelof e a e the liver is of great significance in treatinghypercholesterolemia.

Thls re1ate to a {method of fnducmg a hypo The experimental resultslisted hereafter use methods cholesteFolemlc effect sublects Wifeflngfrom abnor' reported by several prior investigators such as Viviani, R.,t hlgh cholesterol.levels m plasma and to et al., Experientia, 19, 188(1963). The latter investigator Posmons i i g i x j 11 h 1 t l l l alsorecommended the rat as the ideal test animal for th 523 :2 2 g i g ggzggg gg gg ig z gg 2;: correlation with hypocholesterolemic effects in man.The tor to the formation of arteriosclerosis by many medical foilowmgresults are denved from acute test procedure groups such as reported bythe American Heart Associa- P grgups 3% Seven g i g Rgver average tionin 1965 and by various reports of the National Instit a out W ave Fmaqltamed on a tutes of Health. Several chemical compounds have demofStandard rat rat1n (Punna) onstrated cholesterol lowering orhypolipidemic activity i by 011 m whlfih the mammals are such asnicotinic acid, sodium dextrothyroxine, sitosterol, admmlsteredlirapefltoneally dlsSOlVed 111 SallIle- In the cholestyramine andclofibrate. Indications for dextrothytest? PQ 111 Table I, a total ofgtotal roxine and clofibrate, for example, are listed in Modern ammo awas e In e case composed of equlvalent Drugs, 1967, pages 265 and 267.proportions of the individual amino acid. The animals are In the searchfor hypolipidemic agents, it has been resacrificed four hours afteradministration; the individual peatedly reported that sulfur-containingamino acids, such livers and plasma are then assayed and averaged.

TABLE I Plasma cholesterol Liver cholesterol Percent Percent Mg., Arng.,from Mg./ AmgJ from Group percent percent control gm. gm. controlControl 58.35 2.30 Glntamic, theonine, proline,

alanine, glycine 40. 76 -17.59 =--30.1 1.75 -0.55 43.9 Glutarnlc acid49.89 8.47 -14.4 2. 05 -0.25 10.8 Threomne 54.10 -4.25 -7.2 1.99 -o.3143.4 Glutamrethreonrne 47.10 11.25 -19.2 1.83 0.48 -20.8 Glntamlc,threonine, proline 41. 27 I117.08 29.2 1.70 0. "-26.0 Alanlne,p roline52.67 9. 19 -14.8 1.88 0. 27 -12.5

Statistically significant, P=0.5. No'rE.--All at 0.5 mgJkg. of nitrogencontent or 3.6 mg./kg. of amino acid.

TABLE II Plasma cholesterol Liver cholesterol Percent Percent Mg., Amg., from Mg./ A mg./ from Group percent percent control gm. gm. controlControl 5 amino acids: 4.0 rug/kg. of N.. e 58. 0 -13. 0 18.3 2. 81 *O.42 -13 3 amino acids:

4 0 rug/kg 59. 3 10.8 -15. 2 2. 43 0. 83 -25. 4. 2 0 mgJkg 62. 3 12. 38. 7 2. 67 -0. 56 -l7. 3 1.0 mgJkg 63.1 7. 9 11.1 2. 59 0. 64 --19. 80.5 IngJkg 65. 8 5. 2 7. 32 2. 63 -0. 60 18. 6

as taurine, ethionine and especially methionine, reduce excessivecholesterolemia while non-sulfur amino acid was reported to be inactive.Also US. No. 2,965,542 describes the use of five a-amino acids to givehypolipidemic eifects but the use of the amino acid combinationhereafter described is not disclosed. I have now found that acombination of glutamic acid and threonine, two naturally occurringamino acids which are sulfur-free, functions as From these data it isapparent that significant hypolipidemic eifects are found with thecombinations of amino acids previously outlined with positivecholesterol lowering effects range over a wide scale. For practicalpurposes, from about 2-50 preferably about 5-40 mg./ kg. of total aminoacid content, in excess of the normal diet, is a useful range. "Onceagain, the quantity of the amino acid combination is as given in totalacid content with a hypolipidemic agent when administered internally,either equivalent amounts of the individual acids.

In a long-range test the preferred three as well as the five amino acidmixtures gave positive cholesterol lowering activities upon oraladministration of 50 mg./kg. total amino acids in equal proportionstwice a day for days to groups of 8 Carworth rats of average weight 72gins. on a diet of standard rat ration (Purina) supplemented with 10%corn oil. The rats were then sacrificed and their plasma and liverlevels assayed by standard methods.

compositions therefore will be used in addition to the normal proteinintake of the subject and will consist of free amino acids usually inthe naturally occurring isomeric form, not protein polypeptides.Glutamic acid and proline, the other two amino acids of the preferredcombination of this invention, while naturally occurring are classifiedas amino acids which are non-essential to life.

The pharmaceutical dosage units containing the combination of aminoacids may be prepared by standard B Significant effects.

This invention therefore comprises the method of producing hypolipidemic(or hypocholesterolemic) effects in hyperlipidemic (orhypercholesterolemic) animals comprising administering internally,preferably orally, an effective but nontoxic quantity of glutamic acidand threonine. The efiective amounts of the amino acid combination arein excess of the normal diet levels of the subject animal derived fromprotein metabolism. Especially preferred is internal administration of ahypolipidemically effective quantity of glutamic acid, threonine andproline. The method of this invention, for example, may compriseadministration of about 2-50 mg./kg., preferably about 5-40 mg./kg. ofthe total amino acid mixture, for convenience sake often comprised ofsubstantially equivalent amounts of the individual amino acidcomponents. The daily requirements should total about 1-8 grams dailyfor the average weight subject.

In practical terms the two, three or five amino acid mixtures may bemade up in about 100-500 mg., preferably 200-350, dosage units for anaverage subject of 70 kg. weight such as in tablets, capsules, lozenges,troches, or sterile parenteral preparations, for example, in salinesolution. These unit dosages are then administered internally,preferably orally, as many times per day as may be effective withouttoxicity, such as from 2-10 times daily, to a hypercholesterolemicsubject.

The dosage units will contain the active amino acid ingredients usuallyin substantially equivalent amounts optionally combined with a standardpharmaceutical carrier. However, the proportions of ingredients may bevaried for example according to the cost of the ingredients withoutaffecting the biological activity of the mixture by retaining abiologically significant quantity of each of the two essentialingredients, glutamic acid and threonine.

Since the individual ingredients of this invention are naturallyoccurring amino acids, toxicity is not a serious problem. In fact, notoxic side effects were observed in my study. It will be clear to thoseskilled in the art that the hypolipidemically effective amounts of theamino acid combinations of this invention are much greater than thoseoccurring naturally in the diet. For example, the range of normalrequirements for the essential amino acid, threonine, for a normal manderived from protein is 0.30-0.50 g. per day while my experiments showsubstantial added amounts of threonine alone do not have ahypocholesterolemic effect. The claimed amino acid methods inmanufacturing pharmacy. For example, as noted the unit may be a tablet,capsule, lozenge, sublingual tablet, troche, aqueous suspension orsolution or a sterile parenteral solution. Such are prepared by standardpharmaceutical procedures.

EXAMPLE 1 A mixture of 100 mg. each of threonine and glutamic acid ismixed with 100 mg. of lactose, screened and filled into a hard gelatincapsule. These capsules containing 200 mg. of amino acid each are thenadministered orally 6 times daily to a hypercholesterolemic subject ofkg. weight.

EXAMPLE 2 A mixture of 50 mg. each of threonine, glutamic acid andproline are dissolved in saline, passed through a microfilter forsterilization and filled into a sterile ampule. Such are injectedparenterally to a 70 kg. weight hypercholesterolemic subject severaltimes daily.

What is claimed is:

1. The method of producing antilipidemic activity in a hyperlipidemicsubject comprising administering orally to said subject a combination ofsubstantially equal amounts of glutamic acid and threonine in ahypolipidemically effective but nontoxic total quantity of from about2-50 rug/kg. such quantity being in excess of the normal ingestion ofsaid glutamic acid and threonine in the diet of said subject.

2. The method of claim 1 in which proline is added to glutamic acid andthreonine and the three amino acids being present in substantially equalamounts with a total of from about 2-50 mg./kg.

3. The method of claim 1 in which the daily dosage of the combinationused is about 1-8 grams daily.

4. The method of claim 2 in which the total amino acid content of thecombination used is from about 1-8 grams daily.

5. The method of claim 2 in which alanine and glycine are added to thecombination.

References Cited UNITED STATES PATENTS 2,411,897 12/1946 Sahyum 4243l92,965,542 12/ 1960 Castaigne 424-319 3,152,955 10/1964 GoW et a1 424319STANLEY J. FRIEDMAN, Primary Examiner

